PROJECT SUMMARY Alcohol produces rewarding and aversive subjective effects. People with increased sensitivity to the aversive effects of alcohol consume lower levels of alcohol and have lower preference for alcohol. These data suggest that enhancing the aversive effects of alcohol may be a useful strategy to reduce alcohol consumption. Currently, the molecular mechanisms of alcohol aversion are poorly understood. Our preliminary data are the first to implicate the nicotinic acetylcholine receptors (nAChRs) in alcohol aversion. The nAChRs are ligand- gated cation channels that enhance neuronal excitability. We found that systemic injection of a pre-clinical nAChR agonist increases alcohol aversion and decreases alcohol consumption in mice. To begin identifying the neural circuits that are important for nAChR-mediated alcohol aversion, we targeted the rostromedial tegmental nucleus (RMTg), which has been recently implicated in aversion. The RMTg is a GABAergic structure that receives glutamatergic input from the lateral habenula and projects to dopamine neurons in the ventral tegmental area (VTA). Micro-injection of a nAChR agonist into the RMTg and VTA reduced alcohol consumption in mice, implicating nAChRs in this projection. Importantly, our data suggest that targeting nAChRs in an aversive neural circuit can reduce alcohol consumption. Our application addresses critical gaps in knowledge in the neural circuitry and molecular mechanisms of alcohol aversion: Does activation of the RMTg-VTA projection increase alcohol aversion and decrease alcohol consumption? Which nAChR subunits are critical for alcohol aversion and on which neurons are they located? To address these questions, we have designed two specific aims. In AIM 1, we will chemogenetically modulate RMTg GABA and VTA dopamine neuron activity using DREADDs to evaluate the contribution of these neuronal populations to alcohol aversion and alcohol consumption in mice. In AIM 2, we will determine which nAChR subunits are important for alcohol aversion and alcohol consumption, and whether they are located on RMTg GABA or VTA dopamine neurons by reducing the expression of specific nAChR subunits in these neuronal populations using genetically targeted RNA interference in transgenic mice. This project will generate new, fundamental knowledge on the molecular mechanisms of alcohol aversion that will inform drug development efforts for alcohol addiction treatment.